Skip to main content
SpringerLink
Log in

A simple and efficient method for analysis of plant growth regulators: a new tool in the chest to combat recalcitrance in plant tissue culture

  • Original Article
  • Published:
Plant Cell, Tissue and Organ Culture (PCTOC) Aims and scope Submit manuscript

Abstract

This report presents a simple, rapid and accessible validated method for quantification of eight major plant growth regulators (PGR): cytokinins (6-(γ,γ-dimethylallylamino)purine (2-iP), benzylaminopurine (BA) and zeatin), auxin (indole-3-acetic acid; IAA), jasmonic acid (JA), salicylic acid (SA), gibberellic acid (GA3) and abscisic acid (ABA) by liquid chromatography mass spectrometry. This method was tested in eight species including agricultural, ornamental and medicinal species: St. John’s wort, African violet, banana, American elm, tobacco, potato, sweet wormwood, and fennel. The method has good reproducibility and good sensitivity with %RSD (percent relative standard deviation) between 1 and 10% for all matrices and recovery values of 89 to 118% for all analytes. Method detection limits were 50.65 ng/g, 203.4 ng/g, 50.65, ng/g, 50.65 ng/g, 203.4 ng/g, 12.7 ng/g, 193 pg/g and 3.08 ng/g, for SA, IAA, zeatin, JA, GA3, ABA, 2-iP, and BA, respectively. Our results with a range of plant species show that this method represents a simple, low-cost method for analysis of PGRs, and may also serve as an useful starting point for the analysis of other related PGRs, as demonstrated by inclusion of the SA derivative, acetylsalicylic acid, and the JA derivatives: 12-oxo-phytodienoic acid and JA-isoleucine. The efficiency of this method will enable its incorporation into the plant tissue culture work flow and through characterization of endogenous PGR levels, will allow for improved method development for recalcitrant species facilitating fundamental and applied studies in plant morphogenesis, propagation and conservation.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Abbreviations

ABA:

Abscisic acid

ASA:

Acetylsalicylic acid

BA:

6-Benzylaminopurine

2,4-D:

2,4-Dichlorophenoxyacetic acid

GA3:

Gibberellic acid

IAA:

Indole-3-acetic acid

2-iP:

6-(γ,γ-dimethylallylamino)purine

JA:

Jasmonic acid

JA-Ile:

Jasmonic acid isoleucine

MeOH:

Methanol

MS:

Mass spectrometry

OPDA:

12-Oxo-phytodienoic acid

PCIB:

p-Chlorophenoxyisobutyric acid

PGR:

Plant growth regulator

%RSD:

Percent relative standard deviation

SA:

Salicylic acid

SIR:

Single ion recording

SJW:

St. John’s wort

SLV:

Single lab validation

TIBA:

2,3,5-Triiodobenzoic acid

TDZ:

Thidiazuron

UPLC:

Ultra-performance liquid chromatography

References

  • AOAC International (2013) Appendix K: Guidelines for dietary supplements and botanicals. In: Official methods of analysis. AOAC International, Arlington, Appendix K: 1–32

  • Arteca RN (1996) Manipulation of growth and photosynthetic processes by plant growth regulators. In: Plant growth substances. Springer, Boston, pp 240–272

    Google Scholar 

  • Assani A, Chabane D, Foroughi-Wehr B, Wenzel G (2006) An improved protocol for microcallus production and whole plant regeneration from recalcitrant banana protoplasts (Musa spp.). Plant Cell Tissue Organ Cult 85:257–264. doi: 10.1007/s11240-005-9058-y

    Article  Google Scholar 

  • Betz JM, Brown PN, Roman MC (2011) Accuracy, precision, and reliability of chemical measurements in natural products research. Fitoterapia 82:44–52. doi: 10.1016/j.fitote.2010.09.011

    Article  CAS  PubMed  Google Scholar 

  • Bliesner DM (2005) Validating chromatographic methods: a practical guide. Wiley, Hoboken

    Google Scholar 

  • Bonga JM, Klimaszewska KK, von Aderkas P (2010) Recalcitrance in clonal propagation, in particular of conifers. Plant Cell Tissue Organ Cult 100:241–254. doi: 10.1007/s11250-009-9647-2

    Article  Google Scholar 

  • Bu X, Regalado EL, Hamilton SE, Welch CJ (2016) The emergence of low-cost compact mass spectrometry detectors for chromatographic analysis. Trends Anal Chem 82:22–34. doi:10.1016/j.trac.2016.04.025

    Article  CAS  Google Scholar 

  • Cai B-D, Ye E-C, Yuan B-F, Feng Y-Q (2015) Sequential solvent induced phase transition extraction for profiling of endogenous phytohormones in plants by liquid chromatography-mass spectrometry. J Chromatogr B 1004:23–29. doi: 10.1016/j.jchromb.2015.09.031

    Article  CAS  Google Scholar 

  • Cutler AJ, Saleem M, Coffey MA, Loewen MK (1989) Role of oxidative stress in cereal protoplast recalcitrance. Plant Cell Tiss Org Cult 18:113–127. doi:10.1007/BF00033470

    Article  CAS  Google Scholar 

  • Enders TA, Strader LC (2015) Auxin activity: past, present, and future. Am J Bot 102:180–196. doi: 10.3732/ajb.1400285

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gantait S, Sinniah UR, Ali MN, Sahu NC (2015) Gibberellins—a multifaceted hormone in plant growth regulatory network. Curr Protein Pept Sci 16:406–412. doi: 10.2174/1389203716666150440125439

    Article  CAS  PubMed  Google Scholar 

  • Gaspar T, Kevers C, Penel C, Greppin H (1996) Plant hormones and plant growth regulators in plant tissue culture. In Vitro Cell Dev Biol Plant 32:272–289

    Article  CAS  Google Scholar 

  • Hewezi T, Jardinaud F, Alibert G, Kallerhoff J (2003) A new approach for efficient regeneration of a recalcitrant genotype of sunflower (Helianthus annuus) by organogenesis induction on split embryonic axes. Plant Cell Tissue Organ Cult 73:81–86. doi:10.1023/A:1022689229547

    Article  CAS  Google Scholar 

  • Holopainen JK, Blande JD (2012) Molecular plant volatile communication. Adv Exp Med Biol 739:17–31. doi: 10.1007/978-1-4614-1704-0_2

    Article  CAS  PubMed  Google Scholar 

  • Huang G-T, Ma S-L, Bai L-P et al (2012) Signal transduction during cold, salt, and drought stresses in plants. Mol Biol Rep 39:969–987. doi: 10.1007/s11033-011-0823-1

    Article  PubMed  Google Scholar 

  • Hutchinson MJ, Saxena PK (1996) Acetylsalicylic acid enhances and synchronizes thidiazuron-induced somatic embryogenesis in geranium (Pelargonium × hortorum Bailey) tissue cultures. Plant Cell Rep 15:512–515. doi: 10.1007/BF00232984

    Article  CAS  PubMed  Google Scholar 

  • Jones AMP, Shukla MR, Biswas GCG, Saxena PK (2014) Protoplast-to-plant regeneration of American elm (Ulmus americana). Protoplasma 252:925–931. doi: 10.1007/s00709-014-0724-y

    Article  PubMed  Google Scholar 

  • Kaur P, Kothari SL (2004) In vitro culture of kodo millet: anfluence of 2,4-D and picloram in combination with kinetin on callus initiation and regeneration. Plant Cell Tissue Organ Cult 77:73–79. doi: 10.1023/B:TICU.0000016505.20448.44

    Article  CAS  Google Scholar 

  • Li H, Murch SJ, Saxena PK (2000) Thidiazuron-induced de novo shoot organogenesis on seedlings, etiolated hypocotyls and stem segments of Huang-qin. Plant Cell Tissue Organ Cult 62:169–173. doi: 10.1023/A:1006491408762

    Article  CAS  Google Scholar 

  • Li G, Lu S, Wu H et al (2015) Determination of multiple phytohormones in fruits by high-performance liquid chromatography with fluorescence detection using dispersive liquid-liquid microextraction followed by precolumn fluorescent labeling. J Sep Sci 38:187–196. doi: 10.1002/jssc.201401131

    Article  CAS  PubMed  Google Scholar 

  • Mithila J, Hall J, Victor JMR, Saxena P (2003) Thidiazuron induces shoot organogenesis at low concentrations and somatic embryogenesis at high concentrations on leaf and petiole explants of African violet (Saintpaulia ionantha Wendl.). Plant Cell Rep 21:408–414. doi: 10.1007/s00299-002-0544-y

    Article  CAS  PubMed  Google Scholar 

  • Moubayidin L, Di Mambro R, Sabatini S (2009) Cytokinin–auxin crosstalk. Trends Plant Sci 14:1360–1385. doi: 10.1016/j.tplants.2009.06.010

    Article  Google Scholar 

  • Mundhara R, Rashid A (2006) Recalcitrant grain legume Vigna radiata, mung bean made to regenerate on change of hormonal and cultural conditions. Plant Cell Tissue Organ Cult 85:265–270. doi: 10.1007/s11240-005-9061-3

    Article  Google Scholar 

  • Murch SJ, Saxena PK (2001) Molecular fate of thidiazuron and its effects on auxin transport in hypocotyls tissues of Pelargonium × hortorum Bailey. Plant Growth Regul 35:269–275. doi:10.1023/A:10144689059593

    Article  CAS  Google Scholar 

  • Murch SJ, Liu C, Romero RM, Saxena PK (2004) In vitro culture and temporary immersion bioreactor production of Crescentia cujete. Plant Cell Tissue Organ Cult 78:63–68. doi:10.1023/B:TICU.0000020397.01895.3e

    Article  CAS  Google Scholar 

  • Murthy B, Murch SJ, Saxena PK (1998) Thidiazuron: a potent regulator of in vitro plant morphogenesis. In Vitro Cell Dev Biol Plant 34:267–275. doi 10.1007/BF02822732

    Article  CAS  Google Scholar 

  • Murthy HN, Hahn EJ, Paek KY (2008) Recurrent somatic embryogenesis and plant regeneration in Coriandrum sativum L. Sci Hortic 118:168–171. doi: 10.1016/j.scienta.2008.05.037

    Article  CAS  Google Scholar 

  • Nguyen AH, Hodgson LM, Erskine W, Barker SJ (2016) An approach to overcoming regeneration recalcitrance in genetic transformation of lupins and other legumes. Plant Cell Tissue Organ Cult 127:623–635. doi: 10.1007/s11240-016-1087-1

    Article  CAS  Google Scholar 

  • Pan X, Welti R, Wang X (2010) Quantitative analysis of major plant hormones in crude plant extracts by high-performance liquid chromatography-mass spectrometry. Nat Protoc 5:986–992. doi: 10.1038/nprot.2010.37

    Article  CAS  PubMed  Google Scholar 

  • Pliego-Alfaro F, Monsalud MJR, Litz RE, Gray DJ, Moon PA (1996) Effect of abscisic acid, osmolarity and partial desiccation on the development of recalcitrant mango somatic embryos. Plant Cell Tissue Organ Cult 44:63–70. doi: 10.1007/BF00045914

    Article  CAS  Google Scholar 

  • Sanago MH, Murch SJ, Slimmon TY, Krishnaraj S, Saxena PK (1995) Morphoregulatory role of thidiazuron: morphogenesis of root outgrowths in thidiazuron-treated geranium (Pelargonium × hortorum Bailey). Plant Cell Rep 15:205–211. doi: 10.1007/BF00193721

    Article  CAS  PubMed  Google Scholar 

  • Sherif S, Jones A, Shukla MR, Saxena PK (2014) Establishment of invasive and non-invasive reporter systems to investigate American elm–Ophiostoma novo-ulmi interactions. Fungal Genet Biol 71:32–41. doi: 10.1016/j.fgb.2014.08.005

    Article  CAS  PubMed  Google Scholar 

  • Sherif SM, Shukla MR, Murch SJ et al (2016) Simultaneous induction of jasmonic acid and disease-responsive genes signifies tolerance of American elm to Dutch elm disease. Sci Rep 6:21934. doi: 10.1038/srep21934

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Shukla MR, Jones AMP, Sullivan JA et al (2012) In vitro conservation of American elm (Ulmus americana): potential role of auxin metabolism in sustained plant proliferation. Can J For Res 42:686–697. doi: 10.1139/x2012-022

    Article  CAS  Google Scholar 

  • Skoog F, Miller CO (1957) Chemical regulation of growth and organ formation in plant tissues cultured in vitro. Symp Soc Exp Biol 11:118–130

    CAS  PubMed  Google Scholar 

  • Song S, Qi T, Wasternack C, Xie D (2014) Jasmonate signaling and crosstalk with gibberellin and ethylene. Curr Opin Plant Biol 21:112–119. doi: 10.1016/j.pbi.2014.07.005

    Article  CAS  PubMed  Google Scholar 

  • Werner T, Schmülling T (2009) Cytokinin action in plant development. Curr Opin Plant Biol 12:527–538. doi: 10.1016/j.pbi.2009.07.002

    Article  CAS  PubMed  Google Scholar 

  • Xu X, Xie G, He L, Zhang J, Xu X, Qian R, Liang G, L J-H (2013) Differences in oxidative stress, antioxidant systems, and microscopic analysis between regenerating callus-derived protoplasts and recalcitrant leaf mesophyll-derived protoplasts of Citrus reticulata Blanco. Plant Cell Tissue Organ Cult 114:161–169. doi: 10.1007/s11240-013-0312-4

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors gratefully acknowledge the financial support of this work by the National Sciences and Engineering Research Council (NSERC) of Canada [Grant Number 46741] and the Gosling Research Institute for Plant Preservation (GRIPP) [Grant Number 050294].

Author information

Authors and Affiliations

  1. Department of Plant Agriculture, Gosling Research Institute for Plant Preservation, University of Guelph, 50 Stone Rd W, Guelph, ON, N1G 2W1, Canada

    Lauren A. E. Erland, Mukund R. Shukla, W. Broc Glover & Praveen K. Saxena

Authors
  1. Lauren A. E. Erland
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mukund R. Shukla
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. W. Broc Glover
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Praveen K. Saxena
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

LAEE participated in conception and design, data acquisition, analysis and interpretation, MRS participated in conception and design, WBG participated in conception and design and data analysis and PKS participated in conception and design and data interpretation. All authors participated in manuscript preparation and gave final approval of the manuscript.

Corresponding author

Correspondence to Praveen K. Saxena.

Ethics declarations

Conflict of interest

The authors declare no conflict of interest.

Additional information

Communicated by Pamela J. Weathers.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Erland, L.A.E., Shukla, M.R., Glover, W.B. et al. A simple and efficient method for analysis of plant growth regulators: a new tool in the chest to combat recalcitrance in plant tissue culture. Plant Cell Tiss Organ Cult 131, 459–470 (2017). https://doi.org/10.1007/s11240-017-1297-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11240-017-1297-1

Keywords

Search

Navigation